The mechanism of bioactivation of halothane by liver microsomes of rats was investigated by measuring the covalent binding of (14C) and (3H) halothane to microsomal protein and lipid under atmospherees of O2 and N2. Under O2, only the (14C) label of halothane bond appreciably to protein and lipid. In contrast, both labels bound covalently to protein and lipid, when the incubations were performed under N2. These results indicate that at least two mechanisms are involved in the bioactivation of halothane by liver microsomes. In the presence of alpha 2, halothane is likely activated by an oxidative dehalogenation mechanism, as was found previously for CHC13. The activation under low oxygen tension may involve the dehalogenation of halothane to a radical or carbene metabolite.